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Methods and compositions for detecting CNS viruses

Inactive Publication Date: 2009-09-10
QUEST DIAGNOSTICS INVESTMENTS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]In one embodiment, one or more of the first, second, or third reaction products are detected using a probe comprising a fluorescent label. In one embodiment, the probe is an oligonucleotide complementary to the target sequence. In another embodiment, a probe and one of the primers of the first, second, and/or third primer pairs are part of a bi-functional molecule, i.e., a Scorpion™. In pa

Problems solved by technology

CNS viral infection is a very serious clinical condition with high mortality rate and poor clinical outcome.
In immuno-compromised individuals, however, it can pass the blood-brain-barrier and cause CNS infection.
Without treatment, the mortality rate can be as high as 50%, and permanent brain damage often occurs.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Materials and Methods

[0081]In accordance with the methods of the present invention, viral pathogens were detected in biological samples following the procedures described in this example. Using methods similar to the ones described below, it would be possible for the skilled artisan to alter the parameters for the detection of additional target nucleic acids or use alternate probe / primer designs to the ones shown herein.

[0082]The diagnostic panel in this example is designed to detect any of Enterovirus (EV) RNA, Herpes Simplex virus type-1 & -2 (HSV-1 / -2) DNA, or Varicella-Zoster virus (VZV) DNA in a single well reaction. Specifically, it is designed to detect Enterovirus RNA 5′ untranslated region (UTR), HSV-1 and -2 UL29 gene, and VZV gene 36. The assay includes reagents and primers for the simultaneous detection of nucleic acid from the target EV, HSV-1 and -2 and VZV. The assay may further include an internal control (IC) to verify adequate processing of the target viruses and t...

example 2

Enterovirus Serotype Detection Coverage

[0098]A comparison of the genomic sequences from EV serotypes demonstrated that the EV RNA genome is highly polymorphic and there is no identical sequence among all of the serotypes. However, the 5′-UTR contains three very small, but highly conserved regions across most classified EV serotypes (See U.S. Pat. No. 5,075,212). Although it is highly conserved, this region is not a 100% consensus for all EV serotypes. Various mutations have been identified in the region, and mutations in the region could affect the PCR amplification and detection. EV serotypes that contain mutations in the 5′-UTR conserved region were identified (indicated by the * in Table 11). These serotypes were defined as “variable serotypes”.

[0099]To verify the serotype detection coverage for 64 EV serotypes, each serotype was assayed using the methods described in Example 1 (herein “Simplexa™”). All ATCC strains of the “EV serotype panel” were extracted by QIAmp® Viral RNA Mi...

example 3

Sensitivity and Specificity of the Detection Methods of the Invention

[0103]The sensitivity and specificity of the detection methods of the invention (herein “Simplexa™”) were investigated in this Example. A total of 350 CSF specimens were collected from the Focus Reference Lab. These specimens were originally submitted for Enterovirus (EV), Herpes Simplex virus type 1- and / or -2 (HSV-1 / -2) and / or Varicella Zostar Virus (VZV) molecular tests. The majority of the samples were collected during 2004 and stored at −80° C. prior to extraction.

[0104]Due to the limited number of VZV specimens, 22 spiked VZV samples were contrived. VZV viral strain (Strain Ellen Lot 24W) and commercial CSF matrix (Medical Analysis System, Inc. Lot SF07051) were used to create the spiked samples. The spiked samples were made to cover a wide range of viral CSF viral loads with a 2-fold dilution series including 22 samples, starting from 100× ATCC stock dilution. The lower end of the concentration was made to r...

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PUM

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Abstract

The present invention generally relates to a molecular test of enterovirus, herpes simplex virus-1 and -2, and / or Varicella-Zoster virus, in order to identify patients with a viral infection, in particular a viral infection of the central nervous system. Accordingly methods and compositions are disclosed to determine the presence or absence of a viral pathogen in a biological sample comprising, wherein the target nucleic acids comprise the 5′ UTR of the enterovirus genome, UL29 of herpes simplex virus and gene 36 of Varicella-Zoster virus.

Description

FIELD OF THE INVENTION[0001]The present invention relates generally to the field of pathogen detection. In particular, the present invention relates to methods of detecting enterovirus, herpes simplex virus, and Varicella-Zoster viruses in a biological sample.BACKGROUND OF THE INVENTION[0002]The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.[0003]Central nervous system viral infection includes viral encephalitis, aseptic meningitis, meningoencephalitis and myelitis. CNS viral infection is a very serious clinical condition with high mortality rate and poor clinical outcome. There are more than 65,000 CNS viral infections reported annually in the United States. Over 100 viruses are known to cause CNS infections. Among them, Enterovirus (EV), Herpes Simplex Virus type 1 and type 2 (HSV-1 and -2) are the most common and prevalent pathogens causing this...

Claims

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Application Information

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IPC IPC(8): C12Q1/70
CPCC12Q1/705
Inventor CHEN, FANKONG, LILLY I.LEE, MING-CHOUCHEN, JULESTABB, MICHELLE M.AYE, MICHAEL
Owner QUEST DIAGNOSTICS INVESTMENTS INC
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